Global warming increases the surface waters and biodiversity in polar regions. However, the intrinsic biological sources of dissolved organic matter (DOM) in Antarctic surface waters remain poorly understood. This work evaluated the sources and driving mechanisms of DOM in Antarctic lakes systematically, based on fluorescence excitation-emission matrices, ultrahigh-resolution mass spectra, biological detection, and metagenomic analyses. The most abundant DOM in the water was peptides (37.02%), which differed from those in soil (lignins: 26.33%) and penguin guano (lipids: 50.71%). The relative abundance of CHON and CHOP compounds in water was significantly correlated with the distance from the penguin colony (p <
0.05). Both the fluorescence and mass spectrum fingerprints of water and soil/faeces showed low similarities using end-member source tracking methods. This could be attributed to the facilitation of guano-derived nutrients to phytoplankton proliferation, whereas the concentrations of NH